Comparison of fluorescence, laser-induced fluorescence, and ultraviolet absorbance detection for measuring HPLC fractionated protein/peptide mixtures

Proteomics is the study of all proteins in a biological sample. High-pressure liquid chromatography coupled online with mass spectrometry (HPLC/MS) is currently the method of choice for proteomic analysis. Proteins are extracted, separated at the protein or peptide level (after enzymatic digestion), and fractions are analyzed by HPLC/MS. Detection during off-line fractionation is generally conducted using UV-vis, which is not sensitive enough to distinguish fractions having the largest concentration of proteins/peptides and should not be combined prior to HPLC/MS. To overcome this deficiency, we utilize fluorescence or UV-laser induced fluorescence (UV-LIF) detection for measuring proteins/peptides during the off-line fractionation. Fluorescence detection allows low-abundance proteins/peptides that contain aromatic amino acids to be measured. In this study, peptide/protein samples fractionated using ion-exchange chromatography were detected using UV absorbance, fluorescence, and UV-LIF. The results indicated that fluorescence and UV-LIF were able to detect the lower abundance proteins/peptides to give a more representative chromatogram, allowing the analyst to decide which fractions should be combined prior to HPLC/tandem mass spectrometry (MS/MS) analysis.     

Multifunctional encoded self-assembling protein nanofibrils as platform for high-throughput and multiplexed detection of biomolecules

A one-dimensional nanofibrillar array formed by the co-assembly of native and biotin-functionalized beta-amyloid (Aβ) peptide was developed for biomolecule sensing. With the presence of biotin moiety, a variety of biomolecular probes can be conjugated onto the nanofibrils, thus converting the protein assembly into a miniature biosensor. In this work, DNA probes were immobilized onto the fibril for the detection of cDNA sequences. The as-developed "DNA-nanoarray" achieved a detection limit at subattomole level (183 fM in 10 μL). This highly sensitive, yet simple, assay requires a trace amount of sample consumption (<10 μL) and is pretreatment-free. In addition, we reported the preparation of alternate-segmented amyloid nanofibrils with multifunctionality. The fibrils hereby serve as an encoded template that can be visualized with various fluorescence labeling dyes for barcode recognition purpose, and, hence, multiplex detection of biomolecules was achieved. Regarding that each protein nanofibril represents a single detection platform, a large number of single fibrils simultaneously are monitored with the dual-color TIRFM in a high-throughput manner.     

Engineered Amp C β-lactamase as a fluorescent screening tool for class C β-lactamase inhibitors

Class C β-lactamases mediate antibiotic resistance in bacteria by efficiently hydrolyzing a broad range of β-lactam antibiotics. With their clinical significance and the lack of commercially available effective inhibitors, development of class C β-lactamase inhibitors has become one of the recent hot issues in the pharmaceutical industry. In this paper, we report the protein engineering of a fluorescent Amp C β-lactamase mutant designated as V211Cf for the in vitro screening of class C β-lactamase inhibitors. When a fluorescein (f) was incorporated at the entrance of the enzyme's active site (position 211), Amp C β-lactamase from Enterobacter cloacae P99 was tailor-made into a novel fluorescent biosensing protein that could display a fluorescence enhancement upon binding with its β-lactam substrates/inhibitors. With its catalytic activity close to the wild-type level, V211Cf can act as a "natural" fluorescent drug target for screening small binding molecules. In addition, V211Cf can allow specific detection for its active-site binding molecules and discriminate them from nondruglike molecules in the screen. Furthermore, V211Cf is amenable to a high throughput format. Taken together, V211Cf demonstrates the potential as an efficient tool for screening class C β-lactamase inhibitors and facilitates the discovery of therapeutics that can combat the clinically important class C β-lactamases.     

Separation and characterization of metallosupramolecular libraries by ion mobility mass spectrometry

The self-assembly of Zn(II) ions and bis(terpyridine) (tpy) ligands carrying 120° or 180° angles between their metal binding sites was utilized to prepare metallosupramolecular libraries with the connectivity. These combinatorial libraries were separated and characterized by ion mobility mass spectrometry (IM MS) and tandem mass spectrometry (MS(2)). The 180°-angle building blocks generate exclusively linear complexes, which were used as standards to determine the architectures of the assemblies resulting from the 120°-angle ligands. The latter ligand geometry promotes the formation of macrocyclic hexamers, but other n-mers with smaller (n = 5) or larger ring sizes (n = 7-9) were identified as minor products, indicating that the angles in the bis(terpyridine) ligand and within the coordinative tpy-Zn(II)-tpy bonds are not as rigid, as previously believed. Macrocyclic and linear isomers were detected in penta- and heptameric assemblies; in the larger octa- and nonameric assemblies, ring-opened conformers with compact and folded geometries were observed in addition to linear extended and cyclic architectures. IM MS(2) experiments provided strong evidence that the macrocycles present in the libraries were already formed in solution, during the self-assembly process, not by dissociation of larger complexes in the gas phase. The IM MS/MS(2) methods provide a means to analyze, based on size and shape (architecture), supramolecular libraries that are not amenable to liquid chromatography, LC-MS, NMR, and/or X-ray techniques.     

Epitaxial growth and rectification characteristics of double perovskite oxide La2NiMnO6 films on Nb-SrTiO3 single crystal substrates

High-quality thin films of double perovskite La₂NiMnO₆ (LNMO) were epitaxially grown on Nb-doped SrTiO₃ (NSTO) substrates by pulsed laser deposition. The films were found to undergo a ferromagnetic-to-paramagnetic transition at ~ 280 K, which is consistent with the literature report. In the electrical measurements, typical rectifying behavior was observed in the LNMO/NSTO heterojunction. The diffusion voltage (V_D) increases linearly with temperature (T) during cooling until T = 170 K. At T < 170 K, V_D increases at a higher rate and the V_D-T relationship becomes non-linear. A disordered phase related spin polarization was used to understand such behaviors in the heterojunctions.     

Room temperature ferromagnetism in Ni doped In2O3 nanoparticles

In the present work, Ni doped In₂O₃ nanoparticles were prepared using simple co-precipitation method. From the x-ray diffraction analysis it is observed that all samples exhibit single phase polycrystalline nature. All the diffraction lines correspond to the bixbyite type cubic structure. A UV visible analysis reveals that optical band gap decreases from 4.63 to 3.84 eV with Ni doping. DC magnetization measurements reveal that Ni doped In₂O₃ nanoparticles exhibit room temperature ferromagnetism.     

Composition dependence of structural and optical properties of Ba(Zrx,Ti1-x)O3 thin films grown on MgO substrates by pulsed laser deposition

Ba(Zr_x,Ti_1-x)O₃ (BZT) films with Zr concentration ranging from 0 to 40% were grown on MgO single crystal substrates by pulsed laser deposition, and their optical properties in the visible range were systematically characterized. A linear increase in the out-of-plane lattice constant of BZT unit cell with increasing Zr content was detected by X-ray diffraction. The surface morphology was observed by atomic force microscopy and the grain size was shown to increase with Zr concentration. Prism coupling and UV-visible transmission spectroscopy techniques were used to analyze the optical properties of the films. Refractive index between 2.15 and 2.3 was observed at 633 and 1547 nm respectively, which decreased with rising Zr content. The BZT films also possessed large optical band gap energy up to 3.92 eV, which increased with rising Zr content. Quadratic electro-optic effect was observed with electro-optic coefficients between 0.11 and 0.81 × 10^− 18 m²/V², which decreased with Zr concentration. Optical loss was estimated from scattering and absorption, and the absorption coefficient dropped with increasing Zr content at near band gap. The obtained results provide information for the design of BZT thin film-based optical devices.     

Relationship between types of surface shear stress profiles and membrane fouling

Shear stress has been recognized as an important parameter in controlling particle back-transport from membrane surfaces. However, little is known of the relationship between transient shear conditions induced by air sparging and fouling control near membrane surfaces. In this paper, the different types of surface shear stress profiles that had beneficial effects on minimizing reversible surface fouling were examined. The relationship between different statistical shear parameters (e.g. time-averaged shear, standard deviation of shear and amplitude of shear) and fouling control that have been used by others were examined as well. It was found that the fouling rate for membranes subjected to transient shear conditions was lower than for membranes subjected to constant shear conditions. The magnitude, duration and frequency of the shear conditions were found to have an impact on the fouling rate of membranes. It was also found that although some statistical shear parameters could generally be used to relate shear and fouling, they were inadequate to relate surface shear stress to fouling, for all transient shear conditions examined.     

An integrated model for the design of air-cooled chiller plants for commercial buildings

Cooling load calculation is the first step in designing the air-conditioning system of a building. The calculated cooling capacity with appropriate buffer is then used to select the number and size of chillers in the system. N + 1 is a common formula used by designers to size the chiller plants in Hong Kong buildings, where N is the actual number of chillers required and 1 is a redundant chiller provided to ensure reliability. This paper reviews the problem of excess capacity and discusses the risk exposure of chiller systems without redundant chillers. The cooling load profiles of the chiller plants of four medium-sized commercial buildings in Hong Kong are reviewed. The risk exposure of chiller systems without redundant chillers can be minimized by applying risk-based preventive maintenance. The just-in-demand design reduces capital cost of the building and frees up funds for continuous energy measurement and improving the energy efficiency of chiller plant systems. This paper presents a model for designing chiller plants that improves the energy efficiency of the plant in a cost effective and thoughtful manner. It is designed with consideration of the life cycle of the plant and real-time continuous commissioning, monitoring, measurement, comparison and execution for better energy management.     

Performance evaluation and design guidelines for stratum ventilation

This paper evaluates the performance of stratum ventilation systems for small to medium individual offices, open offices, classrooms, and retail shops operated under elevated room temperatures according to governmental guidelines. With proper design, stratum ventilation can maintain a thermally comfortable environment that has a horizontal airflow at head level, a small and reverse temperature gradient between the head and foot levels, and a high air distribution performance index.